ANO4 Antibody

What is ANO4 and where is it primarily expressed?

ANO4, also known as TMEM16D, belongs to the anoctamin family of proteins. It is primarily expressed in the central nervous system and certain endocrine glands, with particularly high expression in the zona glomerulosa (ZG) of the adrenal gland. ANO4 has been linked to various neurological disorders and plays an important role in endocrine function . Studies have confirmed that ANO4 expression in the zona glomerulosa is 23.21-fold upregulated compared to the zona fasciculata, making it one of the most highly expressed genes in ZG . Additionally, ANO4 protein expression is detected in the adrenal medulla, with a predominantly cytoplasmic staining pattern .

How does ANO4 differ functionally from other anoctamins like ANO1?

ANO4 exhibits distinct functional properties from the prototypical anoctamin ANO1:

ANO4 overexpression attenuates calcium-mediated aldosterone secretion and cell proliferation, whereas ANO1 overexpression enhances these processes. In functional assays, ANO4 expression results in low levels of calcium-dependent anion transport compared to the significant plasma membrane chloride channel activity of ANO1 .

What are the validated applications for ANO4 antibodies in research?

The ANO4 antibody (19488-1-AP) has been validated for several experimental applications:

The antibody has successfully detected ANO4 in mouse and rat brain tissue via Western blotting, and has been used in immunohistochemistry applications as reported in scientific publications . It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as the optimal dilution may be sample-dependent .

What are the methodological considerations for immunohistochemical detection of ANO4?

For optimal immunohistochemical detection of ANO4 in tissue sections, researchers should follow these methodological guidelines:

• Use formalin-fixed, paraffin-embedded sections (4 μm thickness).

• Perform antigen retrieval using heat treatment with an appropriate epitope retrieval solution (such as Bond Epitope Retrieval Solution 1) for approximately 20 minutes.

• Apply the primary ANO4 antibody at an optimal working dilution of 1/50.

• Utilize a polymer refine detection kit for visualizing the antigens.

• Include negative controls by omitting primary antibodies to confirm staining specificity.

This protocol has successfully demonstrated cytoplasmic, zona glomerulosa-selective expression of ANO4 protein in adrenal tissue samples . For co-localization studies, researchers can pair ANO4 antibody with anti-CYP11B2 antibody to compare expression patterns, as ANO4 typically shows homogeneous expression in the ZG while CYP11B2 often displays a patchy distribution .

How can ANO4 function be assessed in cellular models?

To assess ANO4 function in cellular models, researchers can implement the following methodological approaches:

• Overexpression studies:

  • Transfect cells (e.g., H295R adrenocortical cells) with ANO4 expression vectors.

  • Confirm overexpression via qPCR (12.6-fold increase in mRNA has been reported) and GFP fluorescence for tagged constructs.

  • Assess downstream effects on:

    • Gene expression (NR4A2, CYP11B2)

    • Basal and stimulated aldosterone secretion

    • Cell proliferation using assays such as XTT

• Silencing experiments:

  • Use siRNA to knockdown ANO4 expression.

  • Verify knockdown efficiency at protein level.

  • Measure changes in NR4A2 and CYP11B2 expression.

  • Assess functional outcomes in aldosterone secretion

• Electrophysiological characterization:

  • Perform whole-cell patch-clamp recordings to measure membrane currents.

  • Apply calcium-elevating agents (ionomycin, ATP) to activate ANO4.

  • Analyze current-voltage relationships and reversal potentials to determine channel properties.

  • Manipulate chloride concentrations to distinguish between cation and anion conductances

These approaches provide comprehensive assessment of ANO4's molecular and functional properties in cellular contexts.

What strategies can be used to investigate ANO4 membrane localization?

Several complementary techniques can be employed to investigate ANO4 membrane localization:

• Confocal microscopy with membrane markers:

  • Co-stain cells with ANO4-GFP fusion proteins and membrane markers like pan-Cadherin.

  • Calculate Pearson's correlation coefficient (PCC) to quantify colocalization.

  • Analyze the subcellular distribution of fluorescence signals

• Domain deletion analysis:

  • Generate truncated ANO4 constructs (e.g., removal of the first transmembrane domain).

  • Assess the impact on membrane localization and channel function.

  • This approach has demonstrated that deletion of the first transmembrane domain (Ano4-1-1150del-GFP) significantly reduces both membrane localization and Ca²⁺-evoked current densities

• Biotinylation assay:

  • Use surface protein biotinylation to specifically label and isolate membrane-localized ANO4.

  • Confirm membrane expression through Western blot analysis of biotinylated fractions

• Calcium-dependent trafficking analysis:

  • Examine whether calcium elevation (e.g., via ionomycin) alters ANO4 surface expression.

  • Note that unlike some ion channels, significant ionomycin-induced increases in surface expression have not been observed for ANO4 in HEK293 or ARPE-19 cells

These methodological approaches provide robust evidence for ANO4's membrane localization and trafficking behavior.

How should discrepancies in ANO4 molecular weight be interpreted in Western blot analysis?

When performing Western blot analysis of ANO4, researchers may observe variations in the detected molecular weight. The calculated molecular weight of ANO4 is 111 kDa, but the observed molecular weight typically ranges from 100-111 kDa . These discrepancies may arise from:

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications can alter protein migration.

• Isoform detection: The antibody recognizes both isoform 1 and isoform 2 of ANO4 , which may have slightly different molecular weights.

• Sample preparation conditions: Denaturing conditions, reducing agents, and buffer compositions can influence protein migration patterns.

• Tissue-specific differences: Processing of ANO4 may vary between tissues (e.g., brain versus adrenal tissue).

To address these issues, researchers should:

• Include appropriate positive controls (mouse or rat brain tissue are validated positive controls )

• Use gradient gels for better resolution of higher molecular weight proteins

• Consider tissue-specific optimization of lysis and sample preparation protocols

• Validate findings with alternative ANO4 antibodies when possible

How can researchers address conflicting data regarding ANO4's channel functionality?

The scientific literature contains evolving understanding of ANO4's channel functionality, with initial classification as a chloride channel giving way to evidence supporting its role as a non-selective cation channel. When encountering conflicting data, researchers should:

• Implement comprehensive electrophysiological characterization:

  • Manipulate ionic conditions to distinguish between anion and cation conductances

  • Assess reversal potentials under defined ionic conditions

  • Apply specific channel blockers to isolate conductance components

• Consider cellular context:

  • Different cell types may express auxiliary proteins that modify channel properties

  • Endogenous versus heterologous expression systems may yield different results

  • The native cellular environment of zona glomerulosa cells may differ from standard expression systems

• Evaluate calcium sensitivity:

  • Determine dose-response relationships for calcium activation

  • Compare calcium sensitivity with established anoctamin family members

• Reconcile functional outcomes with channel activity:

  • ANO4 attenuates calcium-mediated aldosterone secretion, which seems contrary to expected outcomes for a chloride channel in this context

  • Consider how non-selective cation channel activity might better explain ANO4's functional effects

This methodological approach enables researchers to resolve seemingly contradictory data regarding ANO4's channel classification.

What is the significance of ANO4 expression in the central nervous system and its link to neurological disorders?

ANO4 is primarily expressed in the central nervous system alongside its presence in endocrine glands, suggesting important neurophysiological functions . Recent research has linked ANO4 to various neurological disorders , warranting further investigation in several areas:

• Neuronal excitability regulation:

  • As a Ca²⁺-dependent cation channel, ANO4 may modulate neuronal excitability and synaptic transmission

  • Researchers should investigate its role in specific neural circuits through electrophysiological recordings in brain slices or primary neuronal cultures

• Multiple sclerosis connection:

  • ANO4 has been mentioned in gene expression profiling of multiple sclerosis pathology

  • Studies examining ANO4 expression patterns in demyelinating lesions could provide insights into its role in neuroinflammatory processes

• Potential therapeutic targeting:

  • Understanding ANO4's role in neurological disorders may reveal novel therapeutic approaches

  • Developing specific modulators of ANO4 activity could have applications in treating conditions where its dysfunction contributes to pathology

Future research should employ conditional knockout models, neuronal-specific expression systems, and advanced imaging techniques to elucidate ANO4's neurophysiological functions.

How does ANO4's role in regulating aldosterone secretion inform our understanding of primary aldosteronism pathophysiology?

ANO4's selective expression in the zona glomerulosa and its role in attenuating calcium-mediated aldosterone secretion suggest potential implications for primary aldosteronism (PA) pathophysiology:

• Comparative expression analysis:

  • ANO4 expression in zona glomerulosa is 23.21-fold higher than in zona fasciculata, but its expression in aldosterone-producing adenomas (APAs) is similar to zona fasciculata levels

  • This down-regulation in adenomas compared to normal zona glomerulosa suggests a potential role in pathological aldosterone production

• Relationship with CYP11B2:

  • While ANO4 shows homogeneous distribution in zona glomerulosa, CYP11B2 (aldosterone synthase) typically shows patchy expression

  • This lack of correlation between ANO4 and CYP11B2 distribution patterns suggests complex regulatory mechanisms

• Functional implications:

  • ANO4 attenuates calcium-mediated aldosterone secretion, suggesting it may serve as a negative regulator of aldosterone production

  • Loss of this negative regulation in adenomas could contribute to autonomous aldosterone production

• Integration with other zona glomerulosa-specific genes:

  • ANO4 joins other zona glomerulosa-specific genes like DACH1, LGR5, and NEFM that suppress aldosterone secretion and proliferation

  • The coordinated expression of these genes may maintain physiological aldosterone regulation

Future research should explore whether ANO4 genetic variants or mutations occur in primary aldosteronism patients and whether targeted restoration of ANO4 function could offer therapeutic benefits.