CHRNA10 Antibody

What is CHRNA10 and what is its physiological role in auditory function?

CHRNA10 is an ionotropic receptor with a crucial role in the modulation of auditory stimuli. The receptor functions through agonist binding that induces conformational changes, leading to the opening of an ion-conducting channel across the plasma membrane. This channel is permeable to various divalent cations, including calcium, whose influx can activate potassium currents that hyperpolarize the cell membrane .

In the ear specifically, CHRNA10 contributes to:

• Reduction in basilar membrane motion

• Alteration of auditory nerve fiber activity

• Reduction in the range of dynamic hearing

• Protection against acoustic trauma

Research using α10 knockout mice has demonstrated that the α10 subunit is required for normal efferent activation of hair cells and for the development/maintenance of normal olivocochlear synapse structure and function . This indicates that despite the ability of α9 subunits to form functional homomeric receptors in vitro, the α10 subunit is essential for proper functioning in vivo.

What are the key characteristics and epitopes targeted by commercially available CHRNA10 antibodies?

CHRNA10 antibodies vary in their target epitopes and host species:

Most CHRNA10 antibodies target sequences within the N-terminal extracellular domain or the intracellular regions, with the majority being rabbit polyclonal antibodies validated for Western blot applications .

How should CHRNA10 antibodies be optimized for Western blot applications?

Optimization of CHRNA10 antibodies for Western blot requires careful consideration of multiple parameters:

• Dilution factors: Different antibodies require specific dilution ranges:

  • Abcam antibody (ab234767): 1/1000

  • Proteintech antibody (55291-1-AP): 1/200-1/1000

  • Novus Biologicals antibody: 1/200-1/1000

  • Abbexa antibody: 1/1000

• Sample preparation:

  • Cell lines demonstrating positive expression: Jurkat, A549, Raji, and HL-60 cells

  • Protein extraction should maintain the native conformation of the receptor

• Detection systems:

  • Secondary antibody recommendations: Goat polyclonal to rabbit IgG at 1/10000 dilution has shown effectiveness

• Controls:

  • Positive control lysates: Use Jurkat (human T cell leukemia cell line) or A549 (human lung carcinoma cell line)

  • Negative controls: Include antibody pre-absorbed with blocking peptide where available

• Expected band size: The predicted molecular weight is approximately 50 kDa, with observed bands typically between 50-52 kDa

What are the recommended methodologies for immunohistochemistry with CHRNA10 antibodies?

For successful immunohistochemistry using CHRNA10 antibodies:

• Tissue preparation:

  • Paraffin embedding has been validated for human tonsil and thymus tissues

  • Fixation protocols should preserve epitope accessibility

• Antibody dilution:

  • Abcam antibody (ab234767): 1/100 for IHC-P

  • Abbexa antibody: 1/10-1/50 for IHC-P

• Antigen retrieval:

  • Heat-induced epitope retrieval is recommended for formalin-fixed tissues

  • Citrate buffer (pH 6.0) is typically effective

• Detection methods:

  • Polymer-based detection systems provide enhanced sensitivity with minimal background

  • DAB (3,3'-diaminobenzidine) chromogen is commonly used

• Expression patterns:

  • In human tissues, positive staining has been observed in tonsil and thymus tissues

  • Expression in ear tissues (cochlear hair cells) is particularly relevant for functional studies

How does the CHRNA10 subunit interact with CHRNA9 to form functional receptors, and what are the experimental implications?

The interaction between CHRNA10 and CHRNA9 is critical for receptor function:

• Subunit assembly:

  • While α9 subunits can form functional homomeric nAChRs in heterologous expression systems, CHRNA10 does not form functional homomeric channels

  • Coexpression of α9 and α10 subunits results in an approximately 100-fold increase in ACh-gated current amplitude compared to α9 homomers alone

  • The heteromeric α9α10 nAChRs possess distinctive pharmacological and biophysical properties that match native hair cell cholinergic receptors

• Experimental considerations:

  • Studies should account for both subunits when investigating hair cell cholinergic biology

  • In α10-/- mice, OHCs remain minimally responsive to ACh due to residual α9 homomeric receptors, but these are insufficient for normal function

  • A key experimental distinction: nicotine does not activate α9α10 receptors (unlike other nAChR subtypes), serving as a useful pharmacological tool

• Functional differences:

  • Compared to homomeric α9 receptors, heteromeric α9α10 receptors display:

    • Faster and more extensive agonist-mediated desensitization

    • Distinct current-voltage relationships

    • Biphasic responses to changes in extracellular calcium

What experimental models are available for studying CHRNA10 function, and what are their limitations?

Several experimental models provide valuable insights into CHRNA10 function:

• Transgenic mouse models:

  • α10 null-mutant mice (Chrna10-/-) have been engineered using standard procedures

  • These mice have been backcrossed and maintained in homozygous congenic B6.CAST-ahl+ mice

  • Key limitation: Species differences in receptor pharmacology between rodents and humans

• Heterologous expression systems:

  • Xenopus oocytes have been used to express recombinant α9, α10, and α9α10 receptors

  • Cell lines (HEK293, CHO) can also be transfected for receptor expression

  • Limitation: May not fully recapitulate the native cellular environment

• Native tissue preparations:

  • Apical turns of the organ of Corti can be excised for electrophysiological recordings

  • IHCs at P8-9 and OHCs at P10-13 have shown maximal ACh-inducible activity

  • Limitation: Technical difficulty and limited viability of ex vivo preparations

• Experimental readouts:

  • Electrophysiological recordings to assess channel function

  • Quantitative RT-PCR to measure gene expression levels

  • Immunohistochemistry to evaluate protein distribution

  • Limitation: Integration of these diverse datasets requires careful interpretation

How can researchers validate the specificity of CHRNA10 antibodies in their experimental systems?

Validating CHRNA10 antibody specificity requires multiple complementary approaches:

• Peptide competition assays:

  • Pre-incubation of the antibody with the immunizing peptide should abolish specific signals

  • Example: Anti-Nicotinic Acetylcholine Receptor α10 antibody (ANC-010) specificity can be confirmed using the corresponding blocking peptide (BLP-NC010)

• Genetic models:

  • Tissues from Chrna10 knockout mice serve as ideal negative controls

  • Decreased or absent signal in knockout samples validates specificity

• Multiple antibody approach:

  • Using antibodies targeting different epitopes of CHRNA10 should yield consistent results

  • Discrepancies may indicate non-specific binding or isoform recognition

• Correlation with mRNA expression:

  • Combining antibody staining with in situ hybridization or RT-PCR provides multilevel validation

  • SYBR Green-based quantitative RT-PCR has been used to assess gene expression levels in CHRNA10 studies

• Positive and negative tissue controls:

  • Known expression patterns: CHRNA10 is highly expressed in outer hair cells of the ear

  • Western blot analysis of rat and mouse brain lysates has shown specific bands that are eliminated by peptide competition

What are the implications of CHRNA10 mutations and polymorphisms for auditory function and disease?

CHRNA10 genetic variations have significant implications:

• Auditory phenotypes:

  • CHRNA10 null mutations in mice result in abnormal efferent innervation patterns in inner hair cells

  • Loss of α10 is more detrimental to synapse formation than elimination of α9, suggesting complex developmental roles beyond channel function

• Association with tobacco response:

  • SNPs in the CHRNA10 gene have reached experiment-wide empirical significance (p=0.048) in association with subjective responses to tobacco ("dizziness")

  • This implicates CHRNA10 in neurological responses to nicotine, despite the receptor not being directly activated by nicotine

• Neurological disorders:

  • Dysregulation of CHRNA10 has been linked to various neurological disorders including:

    • Alzheimer's disease

    • Parkinson's disease

    • Nicotine addiction

• Research methods for genetic studies:

  • DNA extraction from whole blood or EBV transformed cells

  • Genotyping using custom arrays or Illumina Golden Gate technology

  • Analysis of SNPs in CHRN genes with QC measures including genotype call rates >98%

What controls and experimental design considerations are critical when studying CHRNA10 expression in different tissue types?

Robust experimental design for CHRNA10 studies requires:

What are the methodological differences between detecting CHRNA10 in research versus clinical applications?

Although CHRNA10 antibodies are primarily research tools, understanding methodological differences is important:

• Research applications:

  • Focus on mechanistic understanding of receptor function

  • Multiple experimental approaches (WB, IHC, electrophysiology)

  • Less stringent standardization requirements

  • Various sample types: cell lines, animal tissues, recombinant systems

• Potential clinical relevance:

  • Anti-AChR Alpha10 antibodies have been studied in relation to:

    • Myasthenia gravis diagnosis and pathophysiology

    • Impact on neuromuscular transmission

    • Autoimmune neuromuscular disorders

• Methodological distinctions:

  • Research: Greater flexibility in protocols, exploratory analysis

  • Clinical: Would require standardized protocols, validated antibodies, reproducible results

  • All current commercial antibodies specify "For Research Use Only. Not for use in diagnostic procedures"

• Sample handling differences:

  • Research: Various fixation protocols, storage conditions

  • Clinical applications (if developed): Would require standardized sample collection and processing

  • Storage recommendations for antibodies: -20°C with avoidance of freeze-thaw cycles

How can researchers troubleshoot inconsistent results when using CHRNA10 antibodies across different applications?

When facing inconsistent results:

• Application-specific optimization:

  • Western blotting: Adjust protein loading (10-30μg), blocking conditions, antibody concentration

  • IHC: Modify fixation protocols, antigen retrieval methods, incubation times

  • Different applications may require different antibody concentrations (1:100 for IHC vs. 1:1000 for WB)

• Sample preparation factors:

  • Native vs. denatured conditions: CHRNA10 is a membrane protein with complex structure

  • Buffer composition: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) is commonly used for storage

  • Protein extraction methods may affect epitope availability

• Technical considerations:

  • Reconstitution protocols: Some antibodies ship as lyophilized powder requiring reconstitution with double distilled water

  • Storage conditions: -20°C with proper aliquoting to avoid freeze-thaw cycles

  • Centrifugation before use (10000 X g for 5 min) may improve performance

• Antibody selection based on application:

  • Some antibodies are validated for specific applications:

    • Abcam ab234767: WB, IHC-P

    • Assay Genie CAB3042: Western blot

    • Proteintech 55291-1-AP: WB, ELISA

  • Always verify validation status for your specific application

What considerations are important when studying CHRNA10 in relation to other nicotinic receptor subunits?

Understanding CHRNA10 in the context of other subunits requires:

• Subunit interactions:

  • CHRNA10 operates functionally with CHRNA9 in hair cells

  • All nicotinic acetylcholine receptors are pentamers composed of homologous protein subunits

  • Ten α-subunits (α1–α10), four β-subunits (β1–β4), and γ-, δ-, and ϵ-subunits exist with shared homology

• Experimental design for subunit interactions:

  • Co-immunoprecipitation to detect physical interactions

  • Co-expression studies to assess functional interactions

  • FRET/BRET approaches to study proximity and dynamics

• Pharmacological profiling:

  • α9α10 receptors have distinctive pharmacological profiles distinguishing them from other nAChRs

  • Key distinction: Activation by ACh but lack of activation by nicotine

  • These properties can be exploited experimentally

• Expression pattern analysis:

  • While CHRNA10 is primarily expressed in outer hair cells, other tissues may express different combinations of nAChR subunits

  • Comparative analysis across tissues helps understand subunit-specific functions

  • CHRNA10 expression in the brain suggests potential roles beyond auditory function