Recombinant Mouse Transmembrane protein 151A (Tmem151a)

What is the basic membrane topology of TMEM151A?

TMEM151A exhibits a well-defined transmembrane domain architecture comprising two membrane-spanning alpha helices connected by a short extracellular loop and an intramembrane helix-hinge-helix structure. Recent molecular dynamics simulations, immunocytochemistry, and electron microscopy studies have revealed that most of the protein is oriented toward the intracellular side of cellular membranes. This orientation features a large cytosolic domain with a combination of alpha-helix and beta-sheet structures, as well as the protein's N- and C-termini . This topological organization provides critical structural context for understanding how mutations in specific domains might affect protein function in disease states.

Where is TMEM151A primarily expressed in mice?

TMEM151A is highly expressed in the mouse brain, with particularly strong expression in the cerebral cortex and thalamus . Within neurons, the protein distributes in both axons and dendrites as demonstrated by transfection experiments in primary cortical neurons . Subcellular localization studies have shown that TMEM151A colocalizes with endoplasmic reticulum markers such as Calnexin, suggesting an important role in ER function or ER-associated processes in neuronal cells . This expression pattern aligns with the protein's proposed roles in neurological function and its association with movement disorders.

How conserved is TMEM151A across species?

TMEM151A is highly conserved across species, indicating its fundamental importance in cellular function . This evolutionary conservation extends to both sequence and structural features, suggesting functional constraints on the protein. The high degree of conservation makes mouse models particularly valuable for understanding human TMEM151A-related pathologies, as findings in mouse systems are likely to have translational relevance. Cross-species analyses of TMEM151A variants can provide insights into functionally critical domains and potential therapeutic targets.

What are the recommended protocols for expressing recombinant mouse TMEM151A in cell culture systems?

For optimal expression of recombinant mouse TMEM151A in cell culture systems, researchers should consider the following methodological approach:

• Vector selection: Construct EGFP-tagged TMEM151A plasmids for easy visualization and tracking of the protein in cells.

• Cell lines: Both COS-7 cells (for subcellular localization studies) and primary cortical neurons (for physiological relevance) have been successfully used for TMEM151A expression .

• Transfection parameters: Standard transfection methods yield good expression, but optimization may be necessary depending on the specific experiment.

• Expression verification: Confirm expression through western blotting and fluorescence microscopy.

• Subcellular localization: Use co-immunostaining with organelle markers such as Calnexin (for ER) to determine subcellular distribution .

This methodology has been validated in studies examining both wild-type and mutant forms of TMEM151A, providing a reliable foundation for further investigations.

What approaches are effective for studying TMEM151A protein-protein interactions?

To investigate TMEM151A protein interactions, researchers should implement a multi-faceted approach combining:

• Co-immunoprecipitation (Co-IP): This technique can identify direct binding partners of TMEM151A using either tagged recombinant protein or antibodies against the endogenous protein.

• Proximity labeling methods: BioID or APEX2-based techniques are particularly valuable for mapping the protein interaction landscape of transmembrane proteins like TMEM151A.

• Yeast two-hybrid screening: Modified membrane yeast two-hybrid systems can be employed to discover novel interactors.

• Mass spectrometry analysis: Following immunoprecipitation, mass spectrometry can identify the complement of proteins associated with TMEM151A.

• Validation studies: Confirm interactions through reverse Co-IP, FRET analysis, or GST pull-down assays.

These methodologies should be applied in physiologically relevant contexts, such as neuronal cell lines or primary cultures, to maximize translational relevance of findings.

How can researchers effectively generate and validate TMEM151A mutations that mimic disease-causing variants?

To generate and validate TMEM151A mutations that mimic disease-causing variants, researchers should follow this comprehensive workflow:

• Mutation selection: Target mutations identified in human PKD patients, such as c.140T>C (p.Leu47Pro), c.133T>G (p.Cys45Arg), or c.166G>C (p.Gly56Arg) .

• Mutagenesis method: Use site-directed mutagenesis of wild-type TMEM151A expression plasmids.

• Verification: Confirm mutations through sequencing before proceeding to functional studies.

• Expression analysis: Compare protein expression levels between wild-type and mutant TMEM151A through western blotting and quantitative imaging .

• Subcellular localization: Determine if mutations alter the protein's cellular distribution using immunofluorescence microscopy .

• Functional assays: Develop assays specific to hypothesized TMEM151A functions (e.g., ion channel activity if relevant).

Studies have shown that disease-causing mutations can affect protein expression levels, suggesting that haploinsufficiency might be a pathogenic mechanism for TMEM151A-related disorders .

What electrophysiological methods are most appropriate for investigating potential ion channel properties of TMEM151A?

If TMEM151A functions as an ion channel as hypothesized, the following electrophysiological approaches would be most informative:

• Patch-clamp recordings: Whole-cell, cell-attached, and inside-out configurations should be employed to characterize channel properties in heterologous expression systems (HEK293 or Xenopus oocytes).

• Ion selectivity assays: Systematic ion substitution experiments can determine which ions (Na+, Ca2+, K+, Cl-) might permeate through TMEM151A channels.

• Voltage-dependence analysis: Establish current-voltage relationships across different membrane potentials.

• Pharmacological profiling: Test sensitivity to known ion channel blockers, particularly anticonvulsants like carbamazepine and lamotrigine that effectively treat PKD .

• Mutant analysis: Compare electrophysiological properties of wild-type and disease-associated mutant forms of TMEM151A.

The hypothesized ion channel function is supported by clinical observations that PKD patients with TMEM151A variants respond to carbamazepine treatment, similar to patients with mutations in other ion channel genes .

How can researchers determine if TMEM151A interacts with PRRT2, another protein implicated in paroxysmal kinesigenic dyskinesia?

To investigate potential functional relationships between TMEM151A and PRRT2, implement the following experimental strategy:

• Co-immunoprecipitation studies: Determine whether TMEM151A and PRRT2 physically interact in neuronal cells or heterologous expression systems.

• Co-localization analysis: Use high-resolution microscopy techniques (STED, STORM) to examine spatial relationships between these proteins in neuronal compartments.

• Functional redundancy testing: Compare phenotypes of TMEM151A and PRRT2 knockout models to identify shared and distinct functions.

• Epistasis experiments: Express combinations of wild-type and mutant forms of both proteins to determine functional interactions.

• Shared pathway analysis: Investigate whether both proteins affect common downstream targets such as voltage-gated sodium channels, as PRRT2 is known to modulate Na+ channels .

This investigation is particularly warranted given that both TMEM151A and PRRT2 mutations cause phenotypically similar PKD presentations, suggesting potential convergence in pathogenic mechanisms .

What approaches can be used to generate and characterize mouse models of TMEM151A-associated disorders?

Development of mouse models for TMEM151A-associated disorders should follow this methodological framework:

• Model generation strategies:

  • CRISPR/Cas9-mediated knockin of specific disease mutations (e.g., p.Leu47Pro)

  • Conditional knockout models to study tissue-specific effects

  • Transgenic overexpression of mutant TMEM151A

• Comprehensive phenotyping:

  • Behavioral assessments focused on movement disorders and paroxysmal events

  • EEG recordings to detect abnormal brain activity

  • Video monitoring to capture spontaneous dyskinesia events

  • Pharmacological challenge tests with PKD triggers

• Molecular and cellular characterization:

  • Gene expression profiling in relevant brain regions

  • Protein-protein interaction studies in vivo

  • Electrophysiological recordings from neurons in brain slices

  • Calcium imaging to assess neuronal excitability

• Therapeutic testing:

  • Response to antiepileptic drugs like carbamazepine and lamotrigine

  • Novel compounds targeting hypothesized TMEM151A functions

Mouse models would be particularly valuable given the limitations of in vitro systems for studying complex movement disorders like PKD.

How should researchers interpret conflicting subcellular localization data for TMEM151A?

When confronting contradictory subcellular localization data for TMEM151A, researchers should implement this systematic approach:

• Method comparison:

  • Evaluate differences between antibody-based detection versus tagged protein expression

  • Compare fixation methods, which can significantly affect membrane protein localization

  • Assess overexpression artifacts versus endogenous protein patterns

• Cell type considerations:

  • Heterologous expression systems may show different localization than native neurons

  • Primary neurons may better reflect physiological distribution than cell lines

• Resolution assessment:

  • Standard confocal microscopy may not distinguish between closely associated membranes

  • Super-resolution techniques provide more definitive subcellular assignments

• Integrated analysis framework:

  • Combine biochemical fractionation with imaging studies

  • Use multiple organelle markers simultaneously

  • Consider dynamic trafficking patterns rather than static localization

The observed colocalization with ER markers should be evaluated alongside potential plasma membrane or other organellar associations to develop a comprehensive understanding of TMEM151A's distribution.

What statistical approaches are most appropriate for analyzing expression level changes in TMEM151A mutants?

For rigorous analysis of expression level differences between wild-type and mutant TMEM151A variants, researchers should apply these statistical methodologies:

• Quantification methods:

  • Densitometry analysis of Western blots with normalization to loading controls

  • Intensity measurements from immunofluorescence with spatial normalization

  • Flow cytometry for high-throughput single-cell quantification

• Statistical tests:

  • For normally distributed data: paired t-tests or ANOVA with appropriate post-hoc tests

  • For non-normally distributed data: non-parametric alternatives (Mann-Whitney, Kruskal-Wallis)

  • Account for multiple comparisons when examining multiple mutations

• Sample size determination:

  • Power analysis to calculate appropriate replicate numbers

  • Minimum of three independent biological replicates

• Data presentation:

  • Box-and-whisker plots showing distribution characteristics

  • Individual data points alongside means and standard deviations

• Correlation analyses:

  • Relate expression levels to functional outcomes

  • Compare with clinical severity when data is available

These approaches have revealed that certain PKD-associated mutations lead to decreased TMEM151A protein levels, supporting haploinsufficiency as a potential disease mechanism .

How can researchers distinguish between direct functional effects of TMEM151A mutations versus downstream consequences?

To differentiate between primary and secondary effects of TMEM151A mutations, implement this hierarchical experimental design:

• Temporal analysis of effects:

  • Time-course experiments after induction of mutant expression

  • Identification of earliest detectable changes

• Domain-specific mutations:

  • Systematic mutation of different protein domains

  • Correlation of specific structural alterations with functional outcomes

• Rescue experiments:

  • Complementation studies with wild-type protein

  • Domain-swap experiments to localize functional defects

• Separation of biophysical and cellular effects:

  • In vitro protein folding and stability assays

  • Membrane integration efficiency studies

  • Protein-protein interaction mapping

• Systems biology approach:

  • Network analysis of altered pathways

  • Identification of convergence points across different mutations

This multi-faceted strategy helps establish causality rather than mere association between TMEM151A mutations and observed cellular phenotypes.

What screening methodologies can identify compounds that rescue TMEM151A mutant function?

For developing effective compound screening approaches targeting TMEM151A mutations, researchers should implement this comprehensive workflow:

• Assay development:

  • Cell-based reporters of TMEM151A function or expression

  • High-content imaging for protein localization

  • Electrophysiological readouts if ion channel function is confirmed

• Screening strategy:

  • Primary screen: expression/localization rescue in cell lines

  • Secondary screen: functional rescue in neuronal models

  • Counter-screen: specificity for TMEM151A versus related proteins

• Compound libraries:

  • FDA-approved drug libraries for repositioning opportunities

  • Ion channel modulators based on clinical efficacy of anticonvulsants

  • Chemical chaperones for mutations affecting protein stability

• Validation criteria:

  • Dose-response relationships

  • Structure-activity relationships

  • Mechanistic studies confirming target engagement

• Translational endpoints:

  • Ex vivo testing in patient-derived cells when available

  • In vivo efficacy in mouse models of TMEM151A-related PKD

This approach leverages the observation that PKD patients with TMEM151A mutations respond to treatments like carbamazepine and lamotrigine , suggesting therapeutic modulation of TMEM151A function is clinically achievable.

How can CRISPR-based approaches be optimized for correcting TMEM151A mutations in cellular and animal models?

To maximize the efficiency and specificity of CRISPR-based gene editing for TMEM151A mutations, implement these methodological refinements:

• Guide RNA design:

  • Multiple computational algorithms to identify optimal target sequences

  • Experimental validation of predicted guide efficiency

  • Off-target prediction and verification

• Delivery optimization:

  • For neurons: AAV-based delivery systems with neuron-specific promoters

  • For mouse models: zygote injection versus postnatal delivery comparisons

  • For human cells: nucleofection protocols optimized for neuronal progenitors

• Repair template strategies:

  • Short vs. long homology arms for homology-directed repair

  • Silent mutations in repair templates to prevent re-cutting

  • Selection markers with subsequent removal

• Editing verification:

  • Deep sequencing to quantify editing efficiency

  • Western blotting and immunofluorescence to confirm protein correction

  • Functional assays to demonstrate phenotype rescue

• Specificity assessment:

  • Whole-genome sequencing to detect off-target modifications

  • Transcriptome analysis to identify unintended expression changes

These approaches are particularly relevant for dominant TMEM151A mutations causing PKD, where precise correction of heterozygous mutations could restore normal function .

What experimental approaches can determine whether TMEM151A is a direct target of anticonvulsants effective in PKD?

To establish whether TMEM151A is directly modulated by anticonvulsants that treat PKD, researchers should employ this systematic investigation:

• Direct binding studies:

  • Surface plasmon resonance with purified TMEM151A protein

  • Radioligand binding assays with labeled anticonvulsants

  • Photoaffinity labeling to identify binding sites

• Functional modulation assays:

  • Electrophysiological recordings in the presence of anticonvulsants

  • Dose-response relationships for wild-type versus mutant TMEM151A

  • Kinetic analyses of drug effects

• Structural biology approaches:

  • Cryo-EM studies of TMEM151A in the presence and absence of drugs

  • Molecular docking simulations to predict binding sites

  • Mutagenesis of predicted binding residues to confirm functional importance

• Cellular response patterns:

  • Compare effects of anticonvulsants on cells expressing TMEM151A versus control cells

  • Evaluate drug effects on TMEM151A trafficking and protein-protein interactions

• In vivo correlation studies:

  • Relate drug levels to TMEM151A modulation in animal models

  • Compare pharmacokinetics with pharmacodynamic effects

This research direction is supported by clinical observations that PKD patients with TMEM151A mutations respond well to standard anticonvulsants like carbamazepine and lamotrigine , similar to other ion channel-related neurological disorders.