TRPV4 Antibody

What is the molecular structure and function of TRPV4?

TRPV4 is a tetrameric ion channel with each subunit containing 6 transmembrane domains, a pore-forming loop, and 6 highly conserved ankyrin repeat domains in the cytoplasmic N-terminus . It functions as a non-selective calcium-permeant cation channel involved in osmotic sensitivity and mechanosensitivity . The protein has a theoretical molecular weight of 98.3 kDa, though observed weights can range from 85-102 kDa in experimental conditions due to post-translational modifications .

TRPV4 is activated by multiple stimuli including:

• Thermal stress and heat

• Fatty acid metabolites

• Hypotonicity

• Low pH

• Citrate

• Phorbol esters such as 4α-PDD

Channel activity appears to be regulated by a calmodulin-dependent mechanism with a negative feedback system . TRPV4-mediated calcium influx activates multiple signaling pathways including calcineurin, which promotes nuclear factor of activated T-cells (NFAT) and nuclear factor kappa B (NF-kB) expression .

Where is TRPV4 expressed in mammalian tissues?

TRPV4 demonstrates widespread expression across tissues and cell types:

For immunohistochemical visualization, TRPV4 expression has been confirmed in tissue-resident macrophages located in the lung, gut, brain, liver, and skin . In sweat glands, TRPV4 shows distinct localization in secretory cells and duct cells, with stronger expression in basal and apical membranes .

What applications are TRPV4 antibodies most suitable for?

Based on validation data from multiple sources, TRPV4 antibodies have been successfully employed in the following applications:

When selecting applications, researchers should consult specific product validation data, as performance varies significantly between different antibody preparations .

How should I select the appropriate TRPV4 antibody for my research?

When selecting a TRPV4 antibody, consider these critical factors:

• Target species compatibility: Verify reactivity with your species of interest. Available antibodies show reactivity with human, mouse, rat, and other mammalian species .

• Epitope location: Antibodies target different regions (N-terminal, middle region, C-terminal). For example:

  • Alomone Labs antibody targets residues 853-871 of rat TRPV4 (C-terminus)

  • Novus Biologicals antibody targets the middle region with peptide sequence: RVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPR

  • Cell Signaling antibody targets residues near the carboxy terminus of human TRPV4

• Validated applications: Ensure the antibody has been validated for your specific application. Review validation images provided by manufacturers .

• Controls: Consider availability of blocking peptides for specificity confirmation .

• Clonality: Both monoclonal and polyclonal options are available; polyclonals may provide better sensitivity but potentially less specificity .

For crucial experiments, use multiple antibodies targeting different epitopes to cross-validate results, and always include appropriate positive and negative controls.

What are the optimal methods for detection of TRPV4 by Western blot?

To achieve optimal Western blot results with TRPV4 antibodies:

• Sample preparation:

  • Use fresh tissue/cell lysates when possible

  • Include protease inhibitors to prevent degradation

  • Consider both reduced and non-reduced conditions for complete analysis

• Protocol optimization:

  • Expected molecular weight: 85-102 kDa (theoretical 98 kDa)

  • Recommended dilutions: 1:200 - 1:1000 depending on antibody

  • Consider gradient gels (4-12%) for better resolution

  • Use positive control tissues with known TRPV4 expression (human testis tissue, SK-N-SH cell lysate have been validated)

• Verification strategies:

  • Use blocking peptides where available to confirm specificity

  • Include TRPV4 knockout/knockdown samples as negative controls

  • Heating samples at 37°C rather than 95°C may help preserve membrane protein structure

• Troubleshooting multiple bands:

  • At least six isoforms of TRPV4 are known to exist

  • Post-translational modifications may produce bands of varying molecular weights

  • Non-specific binding can be reduced with optimized blocking (5% non-fat milk or BSA)

The use of TRPV4 knockout tissues or cells provides the most definitive negative control, as demonstrated in validation studies .

What methods are recommended for immunohistochemical detection of TRPV4?

For successful immunohistochemical or immunofluorescence detection of TRPV4:

• Tissue preparation:

  • Both paraffin-embedded and frozen sections have been successfully used

  • For paraformaldehyde-fixed tissues, optimal fixation time is typically 24 hours

  • Antigen retrieval is critical for paraffin sections (heat-induced epitope retrieval in citrate buffer)

• Staining protocol:

  • Starting antibody dilutions: 2-5 μg/mL

  • Incubation: Overnight at 4°C yields best results

  • For fluorescence detection, AlexaFluor secondary antibodies provide superior signal-to-noise ratio

  • Include nuclear counterstain (DAPI or Hoechst) for proper tissue orientation

• Co-localization studies:

  • Combine TRPV4 staining with cell-type specific markers for definitive identification

  • Validated markers for co-staining include:

    • Cytokeratin 8 (CK8) for secretory cells

    • Calcitonin gene-related peptide (CGRP) for specific secretory cell types

    • Parvalbumin for certain neuronal populations

• Controls and validation:

  • Tissue from TRPV4 knockout animals serves as ideal negative control

  • Peptide competition assays confirm specificity

  • Include secondary-antibody-only controls to assess background

For optimal resolution of subcellular localization, super-resolution imaging techniques such as Airyscan imaging have successfully revealed TRPV4 distribution patterns in the basal and apical membranes of cells .

How can functional studies complement antibody-based TRPV4 detection?

Combining antibody-based detection with functional assays provides comprehensive insights into TRPV4 biology:

• Calcium imaging techniques:

  • Use ratiometric calcium indicators (Fura-2) or genetically encoded sensors (GCaMP) to measure TRPV4-mediated calcium influx

  • Selective TRPV4 agonists like GSK1016790A can confirm functional expression

  • Combined with immunostaining, this approach validates that detected protein is functionally active

• Electrophysiological recordings:

  • Patch-clamp recordings can characterize TRPV4 channel properties

  • TRPV4 shows outward rectification when activated by hypotonicity

  • Channel properties can be correlated with protein expression levels determined by immunostaining

• Genetic manipulation approaches:

  • Use TRPV4 knockout models (mice have been extensively validated)

  • TRPV4 siRNA knockdown in cell culture models

  • Correlation of phenotypic changes with altered protein expression levels

• Pharmacological interventions:

  • TRPV4 activation: 4α-PDD (10 μM), GSK1016790A

  • TRPV4 inhibition: GSK2798745 and other antagonists

  • Monitor functional responses with and without TRPV4 modulation

The combination of these approaches with antibody-based detection provides robust validation of TRPV4's role in specific cellular contexts.

What methods are available to study TRPV4 in inflammatory processes?

TRPV4 plays complex roles in inflammation, with evidence for both pro-inflammatory and anti-inflammatory functions . To investigate these roles:

• Macrophage isolation and culture techniques:

  • Isolate primary macrophages from different tissues (lung, brain, liver)

  • Assess TRPV4 expression by flow cytometry and immunostaining

  • Compare expression levels between resident macrophages and recruited populations

• Inflammatory stimulation protocols:

  • LPS stimulation (100 ng/mL) has been validated in multiple studies

  • Measure cytokine responses including IL-6, TNF-α, IL-1β, IL-10

  • Compare responses between wild-type and TRPV4-deficient macrophages

• Mechanistic investigation approaches:

  • Calcium imaging to connect TRPV4 activation to downstream signaling

  • Inhibitors of calcium-dependent pathways (calcineurin inhibitors)

  • Assessment of transcription factor activation (NFAT, NF-kB)

• In vivo inflammation models:

  • Pulmonary infection models using Pseudomonas aeruginosa

  • Matrix stiffness-associated inflammation models

  • Comparison of inflammatory responses between wild-type and TRPV4-knockout mice

These contradictory findings highlight the context-dependent nature of TRPV4 function in inflammation.

How can researchers investigate TRPV4's role in mechanotransduction?

TRPV4's mechanosensitive properties make it an important target in mechanotransduction research:

• Shear stress experimental systems:

  • Parallel plate flow chambers for endothelial cells

  • Measure endothelial Ca2+ responses to flow using calcium imaging

  • Compare responses between wild-type and TRPV4-knockout tissues

• Matrix stiffness modulation:

  • Culture cells on substrates of varying stiffness (soft to rigid)

  • Pathological matrix stiffness (25kPa) has been used to study TRPV4 in fibrosis models

  • Combine with TRPV4 antibody staining to assess changes in localization or expression

• Cell-matrix interaction studies:

  • Focal adhesion visualization using paxillin or vinculin staining

  • Co-staining with TRPV4 antibodies to assess colocalization

  • Analysis of stress fiber remodeling in response to mechanical stimuli

• Signaling pathway investigation:

  • Focus on pathways known to interact with TRPV4 in mechanotransduction:

    • Integrin signaling

    • PI3K pathway

    • Rho/ROCK pathway

    • TGFβ1-induced epithelial-mesenchymal transition

Super-resolution microscopy combined with TRPV4 immunostaining can provide insights into the spatial organization of TRPV4 in mechanosensitive structures.

How can I verify the specificity of TRPV4 antibodies?

Verifying antibody specificity is critical for reliable TRPV4 research:

• Genetic controls:

  • TRPV4 knockout tissues/cells provide the gold standard negative control

  • TRPV4 siRNA knockdown for cell culture experiments

  • Example: Anti-TRPV4 antibody showed labeling in wild-type mice skin but not in TRPV4-deficient mice, confirming specificity

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Should abolish specific staining while non-specific binding remains

  • Example: Western blot analysis of rat brain lysates showed signal elimination when antibody was preincubated with TRPV4 blocking peptide

• Multiple antibody validation:

  • Use antibodies targeting different epitopes

  • Compare staining patterns for consistency

  • Cross-validate with functional assays (calcium imaging with selective agonists)

• Expression pattern verification:

  • Compare observed expression with established patterns in literature

  • TRPV4 should be detectable in known positive tissues (lung, kidney, brain)

  • Subcellular localization should be consistent with membrane protein (cell surface, ER)

Comprehensive validation strategies increase confidence in antibody-based TRPV4 detection for critical research applications.

What challenges arise in analyzing TRPV4 expression in complex tissues?

Complex tissues present several challenges for TRPV4 analysis:

• Heterogeneous cell populations:

  • TRPV4 expression varies between cell types within a tissue

  • Example: In sweat glands, TRPV4 expression differs between secretory cells and duct cells

  • Solution: Use multi-color immunofluorescence with cell-type specific markers

• Isoform-specific detection:

  • At least six isoforms of TRPV4 exist

  • Epitope selection determines which isoforms are detected

  • Solution: Use antibodies targeting conserved regions when studying all isoforms, or isoform-specific antibodies for particular variants

• Post-translational modifications:

  • Modifications may affect antibody binding

  • Channel activity regulation involves mechanisms like phosphorylation

  • Solution: Consider phospho-specific antibodies when studying regulated states

• Quantification challenges:

  • Signal intensity variation across tissue sections

  • Background autofluorescence in certain tissues

  • Solution: Include reference standards, appropriate controls, and careful image acquisition parameters

Single-cell approaches (flow cytometry, single-cell RNA-seq combined with protein analysis) can help resolve heterogeneity issues in complex tissues.

How can researchers reconcile contradictory findings regarding TRPV4 function?

The literature contains seemingly contradictory findings about TRPV4 function, particularly in inflammation . To address these:

• Context-dependent analysis:

  • TRPV4 functions differently depending on:

    • Cell type (different macrophage populations respond differently)

    • Activation stimulus (mechanical vs. chemical vs. thermal)

    • Disease state (acute vs. chronic inflammation)

  • Always specify the exact experimental context when reporting findings

• Temporal dynamics consideration:

  • Initial TRPV4 activation may produce different effects than sustained activation

  • Track responses over time rather than single timepoints

  • Consider calcium signaling dynamics (quick transients vs. sustained elevations)

• Methodological reconciliation:

  • Genetic knockout models may show compensatory mechanisms absent in acute pharmacological inhibition

  • Compare results from different methodological approaches:

    • Genetic models (knockout, knockdown)

    • Pharmacological tools (agonists, antagonists)

    • Different assay systems (in vitro, ex vivo, in vivo)

• Systematic review approach:

  • Construct comprehensive tables comparing:

    • Experimental models

    • Cell types

    • Stimulation protocols

    • Readouts

  • Identify patterns explaining apparent contradictions

Contradictory findings likely reflect the complex and multifaceted roles of TRPV4 across different biological contexts rather than experimental errors.

What emerging technologies can advance TRPV4 research?

Several cutting-edge technologies show promise for TRPV4 research:

• High-throughput and omics approaches:

  • Proteomics to identify TRPV4 interaction partners

  • Phosphoproteomics to map post-translational modifications

  • Transcriptomics to analyze downstream gene expression changes

  • These unbiased approaches can reveal network effects beyond individual pathway analyses

• Advanced imaging techniques:

  • Super-resolution microscopy (STED, STORM, Airyscan) for detailed localization

  • Live-cell calcium imaging combined with optogenetic tools

  • Correlative light and electron microscopy for ultrastructural context

• Functional genomics tools:

  • CRISPR-Cas9 for precise gene editing and tagged endogenous protein

  • Domain-specific mutations to dissect structure-function relationships

  • Cell-type specific conditional knockout models

• Computational modeling:

  • Molecular dynamics simulations of TRPV4 channel gating

  • Systems biology approaches to model TRPV4 in signaling networks

  • Integration of structural biology with functional data

These technologies will help resolve remaining questions about TRPV4's complex roles in health and disease.

What therapeutic implications arise from TRPV4 research?

TRPV4-targeted therapeutics show promise for multiple conditions:

• Current clinical development status:

  • GSK2798745 is in phase II clinical trials

  • Small molecule, orally available inhibitor with low nanomolar potency

  • Limited therapeutic advancement despite extensive research suggests mechanistic complexities

• Potential therapeutic applications:

  • Pulmonary disorders: lung injury, edema (including SARS-CoV-2 complications)

  • Cardiovascular diseases: heart failure, ischemic heart disease

  • Pain conditions

  • Inflammatory disorders

  • Fibrotic diseases

  • Cancer

• Research tools needed for therapeutic development:

  • Antibodies for target engagement studies

  • Biomarker development for patient stratification

  • Improved understanding of cell-type specific effects

• Challenges in therapeutic development:

  • Balancing inhibition of pathological functions while preserving physiological roles

  • Need for tissue-specific targeting approaches

  • Understanding of complex roles in both promoting and resolving inflammation

Further research into TRPV4's contextual functions is needed to advance therapeutic development beyond current limitations.

How can TRPV4 protein complexes be effectively studied?

TRPV4 functions within protein complexes that affect its localization and function:

• Protein-protein interaction methods:

  • Co-immunoprecipitation using validated TRPV4 antibodies

  • Proximity ligation assay for in situ detection of protein interactions

  • FRET/BRET approaches for dynamic interaction studies

  • BioID or APEX2 proximity labeling for identifying interaction partners

• Known interaction partners to investigate:

  • ANO1 (calcium-activated chloride channel)

  • AQP5 (water channel) in secretory contexts

  • PKD2 for formation of mechano- and thermosensitive channels in cilia

  • Calmodulin for calcium-dependent regulation

• Functional complex analysis:

  • Simultaneous calcium and chloride imaging in TRPV4-ANO1 studies

  • Mutation of interaction domains to disrupt specific protein-protein interactions

  • Reconstitution experiments in heterologous expression systems

• Subcellular localization studies:

  • Co-localization analysis with markers for:

    • Plasma membrane domains

    • Cytoskeletal elements

    • Cell-cell junctions

    • Specialized structures (primary cilia, focal adhesions)

Understanding these protein complexes will provide insights into how TRPV4 performs diverse functions across different cellular contexts.