ATP12A Antibody

What is ATP12A and why is it significant in respiratory research?

ATP12A is the ATPase H+/K+ transporting non-gastric alpha2 subunit, a protein primarily localized in the cell membrane. In humans, the canonical protein consists of 1039 amino acid residues with a molecular mass of 115.5 kDa. It functions as a proton pump in airway epithelial cells and plays a critical role in airway surface liquid (ASL) acidification. Its significance in respiratory research stems from its upregulation in cystic fibrosis airways, where it contributes to ASL acidification, potentially favoring bacterial infection and airway obstruction . Additionally, ATP12A has been identified as a member of the Cation transport ATPase (P-type) protein family, with expression primarily in airway epithelial cells, particularly in nonciliated MUC5AC+ cells (goblet cells) .

How does ATP12A differ from gastric H+/K+-ATPase?

ATP12A (also known as HKα2 or ATP1AL1) is the non-gastric isoform of the H+/K+-ATPase, distinct from the gastric H+/K+-ATPase (HKα1). While both proteins function as proton pumps coupled to potassium absorption, they differ in tissue distribution, pharmacological sensitivity, and regulatory mechanisms. ATP12A is expressed in airway epithelial cells, colon, and other non-gastric tissues, whereas the gastric H+/K+-ATPase is predominantly found in parietal cells of the stomach. ATP12A requires ATP1B1 as its β-subunit partner for proper trafficking to the plasma membrane, whereas the gastric H+/K+-ATPase associates with ATP4B . This distinction is methodologically important when designing experiments targeting ATP12A, as researchers must ensure antibody specificity against the non-gastric isoform.

What are the known isoforms of ATP12A and how should researchers account for them?

Up to two different isoforms of ATP12A have been reported in humans . When designing experiments with ATP12A antibodies, researchers must consider which isoform(s) their antibody detects. Methodologically, this requires careful antibody selection with verified epitope information. Researchers should validate antibody specificity through western blotting against recombinant isoforms and tissue samples known to express different isoforms. Additionally, when analyzing experimental results, particularly in quantitative studies, researchers should account for potential isoform-specific expression patterns that might vary across different tissues or disease states. Documenting which isoform(s) are being detected is essential for accurate data interpretation and cross-study comparisons.

How does ATP12A expression change in cystic fibrosis and what mechanisms drive this change?

ATP12A expression is significantly increased in the airways of cystic fibrosis (CF) patients compared to non-CF individuals, particularly in nonciliated cells of the airway epithelium and in submucosal glands . This upregulation appears to be a consequence of bacterial infection and inflammation rather than a direct result of CFTR dysfunction. Methodologically, this finding was established through comparative immunofluorescence studies of bronchial samples from CF and non-CF individuals, with quantification of ATP12A expression showing marked differences in the surface epithelium .

The mechanisms driving increased ATP12A expression include bacterial components and inflammatory cytokines. In vitro studies demonstrate that treating bronchial epithelial cells with bacterial supernatants or IL-4 (a cytokine that induces goblet cell hyperplasia) significantly increases ATP12A expression in nonciliated cells . This upregulation is associated with the concomitant upregulation and translocation of ATP1B1 protein from the basal to apical epithelial side, where it colocalizes with ATP12A, facilitating its function . These findings suggest that ATP12A upregulation may represent a detrimental response in CF that further exacerbates lung pathology by promoting airway acidification.

What are the methodological considerations when studying ATP12A trafficking to the plasma membrane?

Studying ATP12A trafficking requires understanding its partnership with beta subunits. Research has conclusively demonstrated that ATP1B1 is the primary partner required for ATP12A trafficking to the plasma membrane . When designing experiments to study this process, researchers should consider:

• Co-expression systems: In heterologous expression systems, co-transfection with ATP1B1 is necessary to move ATP12A from intracellular compartments to the cell surface.

• Visualization techniques: Confocal microscopy with dual immunofluorescence labeling for both ATP12A and ATP1B1 is essential to track their colocalization and trafficking.

• Regulatory influences: IL-4 treatment significantly enhances ATP12A and ATP1B1 colocalization at the apical membrane, making it a useful experimental condition to study trafficking dynamics .

• Subcellular fractionation: When biochemically analyzing ATP12A localization, proper separation of membrane fractions is crucial, with verification using established membrane markers.

These methodological approaches enable researchers to accurately assess the trafficking mechanisms and regulatory factors affecting ATP12A surface expression, which is essential for understanding its function in normal and pathological conditions.

How does ATP12A functionally interact with CFTR-dependent bicarbonate transport?

ATP12A functions as a proton pump that can contribute to acidification of the airway surface liquid (ASL), while CFTR facilitates bicarbonate secretion that alkalinizes the ASL. In non-CF airways, CFTR-dependent bicarbonate transport counterbalances ATP12A-mediated proton secretion, maintaining optimal ASL pH. In CF airways, the absence of functional CFTR leads to impaired bicarbonate transport, allowing unopposed ATP12A activity to acidify the ASL .

Methodologically, when studying this interaction, researchers should:

• Use pH-sensitive fluorescent dyes to measure ASL pH under various conditions.

• Implement ion substitution experiments to distinguish between proton and bicarbonate effects.

• Apply specific inhibitors (ouabain for ATP12A; CFTRinh-172 for CFTR) to dissect the relative contributions of each transport system.

• Consider the use of air-liquid interface cultures that better recapitulate the in vivo airway environment.

Research has shown that inhibiting ATP12A with ouabain can minimize the abnormal acidification observed in CF epithelia, suggesting a therapeutic potential . These methodological approaches allow researchers to evaluate the complex interplay between ATP12A and CFTR in regulating ASL composition and its implications for respiratory health and disease.

What are the optimal conditions for detecting ATP12A in different tissue samples?

Detecting ATP12A in tissue samples requires optimized protocols depending on the application. Based on published methodologies:

For immunofluorescence detection in paraffin-embedded bronchial samples:

• Perform antigen retrieval with 10 mM citrate buffer (pH 6) heated to 95°C for 5 minutes.

• Permeabilize with 0.3% Triton X-100 in PBS for 5 minutes.

• Block with 1% BSA in PBS for 2 hours.

• Incubate with rabbit anti-ATP12A antibody (HPA039526, MilliporeSigma) at 1:400 dilution overnight at 4°C.

• Use goat anti-rabbit Alexa Fluor 488 as a secondary antibody at 1:200 dilution .

For Western blot analysis:

• Lyse cells in RIPA buffer containing protease inhibitors.

• Separate 20 μg of total protein on 4-15% gradient gels.

• Transfer to PVDF membrane.

• Incubate with rabbit anti-ATP12A antibody (HPA039526, MilliporeSigma) at 1:4,000 dilution.

• Use anti-rabbit HRP secondary antibody at 1:50,000 dilution .

These methodological details are critical for reproducible detection of ATP12A across different experimental systems and sample types.

What controls should be included when validating ATP12A antibody specificity?

Validating ATP12A antibody specificity requires a comprehensive set of controls:

• Positive tissue controls: Include airway epithelial cells known to express ATP12A, particularly goblet cells that show high expression levels .

• Negative tissue controls: Include tissues known not to express ATP12A or use samples from ATP12A knockout models if available.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to demonstrate binding specificity.

• Parallel antibody validation: Use multiple antibodies targeting different epitopes of ATP12A to confirm signal concordance.

• siRNA knockdown: Include samples from cells with ATP12A knockdown to verify signal reduction.

• Recombinant protein: Use purified ATP12A protein as a positive control for Western blotting.

• Isotype controls: Include matched isotype antibodies to control for non-specific binding.

• Cross-reactivity assessment: Test antibody against related proteins (e.g., other P-type ATPases) to ensure specificity.

What is the recommended approach for co-localization studies of ATP12A with other epithelial markers?

Co-localization studies of ATP12A with other epithelial markers require careful methodological consideration:

• Multi-channel immunofluorescence protocol:

  • Use primary antibodies from different host species to avoid cross-reactivity.

  • For bronchial epithelium, combine rabbit anti-ATP12A (1:400) with mouse IgG1 anti-MUC5AC (1:200) for goblet cells and mouse IgG2B anti-acetylated tubulin (1:300) for ciliated cells .

  • For ATP12A/ATP1B1 co-localization, combine rabbit anti-ATP12A with mouse IgG2A anti-ATP1B1 (1:1,000) .

  • Use isotype-specific secondary antibodies with minimal spectral overlap.

• Image acquisition considerations:

  • Employ confocal microscopy to minimize out-of-focus signal.

  • Collect sequential scans to prevent bleed-through.

  • Use appropriate negative controls for autofluorescence assessment.

  • Include single-stained samples for channel compensation.

• Quantitative co-localization analysis:

  • Calculate Pearson's or Mander's coefficients for objective assessment.

  • Analyze multiple fields and biological replicates.

  • Apply threshold consistently across all samples.

This methodological approach has successfully demonstrated that ATP12A is predominantly expressed in nonciliated MUC5AC+ cells and co-localizes with ATP1B1 at the apical membrane, particularly after IL-4 treatment .

How should researchers interpret ATP12A expression differences between normal and disease states?

Interpreting ATP12A expression differences requires careful methodological consideration of multiple factors:

• Context-specific evaluation:

  • ATP12A expression is markedly higher in CF airways compared to non-CF airways, but this difference is primarily in the surface epithelium and less dramatic in submucosal glands .

  • Note that some non-CF individuals with bacterial infections (e.g., patient HBE4 with IgG deficiency) may show ATP12A expression comparable to CF patients .

• Multicausal analysis:

  • Enhanced ATP12A signal in disease states may result from both increased goblet cell numbers and increased expression within individual cells.

  • Consider whether expression changes are direct consequences of the disease or secondary responses to infection/inflammation.

• Analytical approach:

  • Quantify ATP12A expression using standardized parameters (fluorescence intensity, Western blot densitometry).

  • Normalize to appropriate housekeeping genes/proteins.

  • Correlate expression with clinical parameters and microbiological data.

  • Consider temporal dynamics in disease progression.

• Interpretation pitfalls to avoid:

  • Don't assume expression differences seen in vivo will be replicated in vitro without appropriate stimuli.

  • Recognize that chronic disease samples may reflect advanced disease states, not early pathogenesis.

  • Consider ATP12A expression in relation to other ion transport proteins for comprehensive interpretation.

These methodological considerations enable accurate interpretation of ATP12A expression data in comparative studies between normal and disease states.

What functional assays can effectively measure ATP12A activity, and how should the data be analyzed?

Functional assessment of ATP12A activity requires specialized assays and careful data analysis:

• pH measurement of apical fluid:

  • Methodology: Measure pH in the apical fluid of cultured epithelia using pH-sensitive fluorescent dyes or microelectrodes.

  • Experimental conditions: Compare pH values under bicarbonate-free vs. bicarbonate-containing conditions to isolate ATP12A contribution.

  • Analysis approach: Calculate rate of acidification and compare between conditions with and without ATP12A inhibition (e.g., ouabain) .

• Rubidium-86 uptake assay:

  • Methodology: Measure Rb+ uptake as a K+ surrogate to assess ATP12A-mediated K+ transport.

  • Analysis: Calculate ouabain-sensitive component of uptake, normalizing to total protein.

• ATPase activity assay:

  • Methodology: Measure inorganic phosphate release from ATP in membrane preparations.

  • Analysis: Calculate the ouabain-sensitive component to isolate ATP12A activity from other ATPases.

• Electrophysiological measurements:

  • Methodology: Use Ussing chambers to measure short-circuit current with ion substitution protocols.

  • Analysis: Identify ATP12A-mediated transport by specific inhibitor sensitivity patterns.

• Data interpretation considerations:

  • Account for compensatory mechanisms and parallel ion transport pathways.

  • Consider the relative contribution of ATP12A vs. other acidification/alkalinization mechanisms.

  • Correlate functional data with protein expression levels.

These methodological approaches allow researchers to effectively measure and interpret ATP12A activity in different experimental systems, providing insights into its physiological and pathophysiological roles.

How can researchers distinguish between ATP12A-mediated effects and other ion transport mechanisms?

Distinguishing ATP12A-mediated effects from other ion transport mechanisms requires systematic methodological approaches:

• Pharmacological dissection:

  • Apply ouabain at concentrations specific for ATP12A inhibition (100 μM-1 mM range).

  • Use CFTRinh-172 to block CFTR-mediated bicarbonate transport.

  • Apply bafilomycin A1 to inhibit V-type H+-ATPases.

  • Use S0859 to inhibit Na+/HCO3- cotransporters.

  • Design experiments with sequential addition of inhibitors to isolate specific contributions.

• Ion substitution experiments:

  • Remove K+ to specifically impair ATP12A function without affecting other transporters.

  • Conduct experiments in bicarbonate-free conditions to eliminate bicarbonate transport contributions.

  • Replace Na+ with NMDG+ to eliminate Na+-dependent processes.

• Genetic manipulation:

  • Use siRNA/shRNA knockdown specific to ATP12A.

  • Implement CRISPR/Cas9 gene editing to create ATP12A knockout models.

  • Express dominant-negative ATP12A mutants.

• Analytical approach:

  • Establish full inhibitor dose-response curves.

  • Calculate additive versus non-additive effects of multiple inhibitors.

  • Apply mathematical modeling to dissect relative contributions.

  • Consider kinetic parameters of transport processes.

• Data interpretation framework:

  • Under bicarbonate-free conditions, rapid acidification indicates ATP12A activity.

  • Ouabain-sensitive acidification and K+ absorption specifically implicate ATP12A.

  • Different cell types may exhibit different proportional contributions of various transport mechanisms.

These methodological approaches enable researchers to specifically attribute observed physiological effects to ATP12A versus other ion transport mechanisms, which is essential for accurately understanding its role in health and disease.

What are the recommended approaches for studying ATP12A expression in response to inflammatory mediators?

Studying ATP12A expression in response to inflammatory mediators requires systematic methodological approaches:

Research has demonstrated that IL-4 treatment induces a strong increase in ATP12A expression, particularly in nonciliated cells, while bacterial supernatants also induce ATP12A expression in both CF and non-CF cells, although to a lesser extent than IL-4 . These findings suggest that ATP12A upregulation may be part of the epithelial response to infection and inflammation, potentially contributing to disease pathogenesis in CF.

What methodological approaches are effective for studying ATP12A in submucosal glands?

Studying ATP12A in submucosal glands presents unique challenges requiring specialized methodologies:

• Tissue preparation and analysis:

  • Collect samples containing intact submucosal glands from bronchial tissue.

  • Use standard fixation (10% neutral buffered formalin) for immunofluorescence studies.

  • Section tissues to visualize both gland acini and ducts.

  • Apply antigen retrieval with 10 mM citrate buffer (pH 6) heated to 95°C .

  • Use confocal microscopy for high-resolution imaging of glandular structures.

• Cell-type identification strategy:

  • Co-stain with markers of different gland cell types (lysozyme for serous cells, MUC5B for mucous cells).

  • Include ductal markers (keratin 7) to distinguish ductal from acinar expression.

  • Use epithelial markers (E-cadherin) to define glandular architecture.

• Functional assessment:

  • Consider ex vivo gland secretion assays with optical measurement of secreted bubbles.

  • Apply pharmacological agents (carbachol, forskolin, ouabain) to manipulate and assess ATP12A function.

  • Collect glandular secretions for pH measurement and compositional analysis.

Research has revealed ATP12A expression in the lumen of submucosal glands, a finding not previously reported . Interestingly, the difference in ATP12A expression between CF and non-CF samples was less dramatic in submucosal glands compared to the surface epithelium, with some non-CF patients displaying comparable ATP12A expression levels to CF patients . This methodological approach to studying ATP12A in submucosal glands provides insights into its potential role in modulating glandular secretions, which may have implications for airway hydration and antimicrobial defense.

What challenges exist in translating ATP12A research findings from in vitro models to clinical applications?

Translating ATP12A research findings from in vitro models to clinical applications faces several methodological challenges:

• Model system limitations:

  • Cell culture systems may not fully recapitulate the complex cellular composition of native airways.

  • Air-liquid interface cultures lack submucosal glands and immune cell interactions.

  • Consider using more complex models (organoids, tissue explants) for validation.

  • Animal models may have species-specific differences in ATP12A expression and function.

• Translation challenges:

  • Inhibitor specificity: Ouabain targets other ATPases at higher concentrations.

  • Delivery challenges: Targeting inhaled inhibitors specifically to ATP12A-expressing cells.

  • Timing considerations: Determining when in disease progression ATP12A inhibition would be most beneficial.

  • Combined therapies: Assessing how ATP12A inhibition interacts with CFTR modulators or antibiotics.

• Methodological approaches to address these challenges:

  • Validate findings across multiple model systems of increasing complexity.

  • Test potential therapies in primary cells from diverse patient populations.

  • Develop ATP12A-specific inhibitors with appropriate pharmacokinetic properties.

  • Design ex vivo studies using freshly explanted human tissues.

  • Consider in vivo imaging techniques to monitor ATP12A activity in animal models.

• Clinical translation roadmap:

  • Focus initial clinical applications on scenarios with established ATP12A upregulation (e.g., CF during exacerbations).

  • Develop biomarkers to identify patients most likely to benefit from ATP12A-targeted therapies.

  • Consider combination approaches targeting both ATP12A and complementary pathways.

What are the key applications and detection methods for ATP12A antibodies?

This comparative table provides methodological guidance for selecting appropriate applications and detection methods for ATP12A antibodies based on research requirements and sample types.

How does ATP12A expression compare across different respiratory conditions?

IF = Immunofluorescence; WB = Western Blot

This comparative table synthesizes research findings on ATP12A expression patterns across different respiratory conditions, highlighting the relationship between bacterial infection, inflammation, and ATP12A upregulation. This information is methodologically valuable for researchers studying ATP12A in the context of respiratory diseases.

What is the relationship between ATP12A, ATP1B1, and functional pH regulation?

This data table illustrates the functional relationship between ATP12A expression, ATP1B1 localization, and their impact on airway surface liquid pH regulation. The table highlights the methodological importance of considering both expression levels and protein trafficking when interpreting functional data related to ATP12A activity in health and disease.