HTR3E Antibody

What is HTR3E and what is its function in human physiology?

HTR3E encodes the subunit E of the type 3 receptor for 5-hydroxytryptamine (serotonin), which functions as a neurotransmitter, hormone, and mitogen. It belongs to the ligand-gated ion channel receptor superfamily and causes fast, depolarizing responses in neurons after activation . The receptor plays critical roles in neuronal signaling and gastrointestinal function. Genes encoding subunits C, D, and E form a cluster on chromosome 3, and alternative splice variants have been described, though full-length sequences for some variants remain undetermined .

Where is HTR3E predominantly expressed in human tissues?

HTR3E is robustly expressed throughout the gastrointestinal tract, particularly in the colon and intestine . Research examining expression across multiple GI regions (jejunum, ileum, colon, sigmoid colon) found that HTR3E was the only one among related receptor genes (compared to HTR3A, HTR3B, and HTR3C) to be consistently and robustly expressed across all these regions . This expression pattern suggests a significant role for HTR3E in gastrointestinal physiology and potentially in GI disorders.

What are the common aliases and designations for HTR3E in scientific literature?

HTR3E is known by several aliases in scientific literature and databases:

Protein Aliases:

• 5-HT3-E

• 5-hydroxytryptamine (serotonin) receptor 3, family member E

• 5-hydroxytryptamine (serotonin) receptor 3E, ionotropic

• 5-hydroxytryptamine receptor 3 subunit E

• 5-hydroxytryptamine receptor 3E

• Serotonin receptor 3E

Gene Aliases:

• 5-HT3-E

• 5-HT3c1

• 5-HT3E

• HTR3E

• MGC120035, MGC120036, MGC120037

Additional identifiers include:

• UniProt ID: A5X5Y0 (Human)

• Entrez Gene ID: 285242 (Human)

What are the primary research applications for HTR3E antibodies?

HTR3E antibodies are utilized in multiple experimental techniques:

• Western Blotting (WB): For detection of denatured HTR3E protein in tissue and cell lysates

• Immunohistochemistry (IHC): For visualization of HTR3E in paraffin-embedded or frozen tissue sections

• Flow Cytometry (FACS): For detection of HTR3E in cell populations

• Enzyme Immunoassay (EIA/ELISA): For quantitative measurement of HTR3E levels

• Immunofluorescence (IF/ICC): For subcellular localization studies

The selection of application depends on research objectives, with different antibodies optimized for specific techniques as indicated in product documentation.

What are important considerations when selecting an HTR3E antibody for experimental use?

When selecting an HTR3E antibody, researchers should consider:

• Binding specificity/epitope: Different antibodies target different regions of HTR3E:

  • Middle region (AA 133-162)

  • Central region (AA 126-154)

  • N-terminal region (AA 26-248)

  • C-terminal region (AA 360-409)

• Host species and clonality: Most HTR3E antibodies are rabbit polyclonal

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, FACS, etc.)

• Species reactivity: While primarily reactive with human HTR3E, some antibodies show cross-reactivity with other species:

  • Human only

  • Human, Dog

  • Human, Dog, Rabbit, Horse

• Immunogen sequence: Some antibodies are raised against specific sequences:

  • "LAFILSRATPRPALGPLSYREHRVALLHLTHSMSTTGRGVTFTINCSGF"

  • KLH-conjugated synthetic peptides from specific regions

What validation methods should researchers employ to confirm HTR3E antibody specificity?

To validate HTR3E antibody specificity:

• Positive controls: Use tissues known to express HTR3E (colon and intestinal samples)

• Negative controls:

  • Primary antibody omission

  • Use of tissues/cells with confirmed low HTR3E expression

  • Blocking peptide competition assays

• Molecular weight verification: Confirm detection at the expected molecular weight of HTR3E (approximately 51 kDa)

• Cross-validation: Compare results using antibodies targeting different epitopes of HTR3E

• Genetic validation: Use HTR3E knockout or knockdown models as negative controls. Commercial CRISPR/Cas9 knockout plasmids for HTR3E are available for this purpose

How is HTR3E implicated in gastrointestinal disorders, particularly IBS-D?

HTR3E has been significantly associated with diarrhea-predominant irritable bowel syndrome (IBS-D), particularly in females. Key findings include:

• Genetic association: Meta-analysis of 2,682 IBS patients and 9,650 controls from 14 international cohorts confirmed that the SNP rs56109847 (also known as rs62625044) in the 3'-UTR of HTR3E is associated with female IBS-D with an odds ratio of 1.58 (95% CI: 1.18-2.12) .

• Expression changes: HTR3E transcript levels were significantly reduced in the sigmoid colon of IBS patients (p = 0.0187), particularly in those with IBS-D (p = 0.0145) . This suggests altered HTR3E expression may contribute to IBS-D pathophysiology.

• Regulatory mechanism: The rs56109847 polymorphism is located in a microRNA binding site (miR-510) in the 3'-UTR of HTR3E. This SNP disrupts miRNA binding, leading to upregulation of HTR3E expression . Experimental data showed:

  • Decreased miR-510 expression in colonic mucosa of IBS-D patients

  • Increased HTR3E expression in patients with the GA genotype compared to the GG genotype

  • The polymorphism significantly upregulated luciferase expression in cellular models

These findings emphasize HTR3E's role in IBS-D pathogenesis and highlight it as a potential therapeutic target.

What experimental approaches can be used to study HTR3E gene variants and their functional effects?

To investigate HTR3E variants and their functional impacts, researchers can employ:

• Genotyping methods:

  • PCR amplification and restriction fragment length polymorphism (RFLP) analysis using specific enzymes (e.g., Hpy188III for rs56109847)

  • Direct sequencing of PCR products

  • PCR protocols often include: incubation at 94°C for 2 minutes; multiple cycles at different annealing temperatures; and final extension at 72°C for 5 minutes

• Expression analysis:

  • qRT-PCR for mRNA quantification in tissues

  • Immunohistochemistry for protein localization and semi-quantitative analysis

  • Western blotting for protein quantification

• Functional assays:

  • Dual-luciferase reporter assays to assess the impact of 3'-UTR variants on gene expression

  • miRNA binding studies using constructs containing wild-type or variant HTR3E 3'-UTR sequences

  • Cell culture models with HTR3E overexpression or knockdown

• Cloning and mutagenesis approaches:

  • Creation of recombinant DNA constructs containing HTR3E 3'-UTR variants

  • Site-directed mutagenesis to introduce specific polymorphisms

  • Transfection of constructs into relevant cell lines (HEK293, HT-29)

What are the latest approaches for manipulating HTR3E expression in experimental models?

Modern molecular tools for manipulating HTR3E expression include:

• CRISPR/Cas9 gene editing:

  • HTR3E CRISPR/Cas9 knockout plasmids (e.g., sc-418317) for complete gene knockout

  • HTR3E HDR plasmids (e.g., sc-418317-HDR) for homology-directed repair and precise gene modification

  • HTR3E Double Nickase plasmids (e.g., sc-418317-NIC) for improved specificity in gene knockout

• CRISPR activation systems:

  • HTR3E CRISPR activation plasmids for enhancing endogenous HTR3E expression

  • Lentiviral activation particles for stable expression modification

• Cell line models:

  • Transfection of HTR3E expression constructs

  • siRNA or shRNA for transient or stable knockdown

  • Selection of appropriate cell lines (intestinal epithelial cells, neuronal cell lines) relevant to HTR3E function

• In vivo models:

  • Transgenic mouse models with altered HTR3E expression

  • Note that mouse HTR3E shows only 33% sequence identity with human HTR3E, potentially limiting translational relevance

What protocols yield optimal results when using HTR3E antibodies for immunohistochemistry?

For optimal immunohistochemical detection of HTR3E:

• Tissue preparation:

  • Formalin-fixed, paraffin-embedded sections or frozen sections of intestinal/colonic tissue

  • Antigen retrieval methods (heat-induced or enzymatic) to expose epitopes

• Antibody dilutions:

  • Typically 1:20 to 1:50 dilution is recommended for IHC applications

  • Optimal dilutions should be determined by the end user through titration experiments

• Detection systems:

  • Polymer-based detection systems for enhanced sensitivity

  • Chromogenic (DAB) or fluorescent-labeled secondary antibodies

• Controls:

  • Positive control: colon tissue (HTR3E is abundantly expressed around colonic mucosal glands)

  • Negative control: omission of primary antibody

• Signal interpretation:

  • HTR3E is predominantly expressed around colonic mucosal glands with less expression in the stroma

  • Compare staining patterns between control and experimental tissues

What are the critical parameters for successful Western blot detection of HTR3E?

For effective Western blot detection of HTR3E:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for tissue/cell lysis

  • Include phosphatase inhibitors if studying phosphorylated forms (HTR3E has a known phosphorylation site at S228)

• Protein loading:

  • Load 20-50 μg of total protein per lane

  • Use intestinal or colonic tissue lysates as positive controls

• Electrophoresis conditions:

  • SDS-PAGE using 10-12% polyacrylamide gels

  • Include molecular weight markers to confirm the expected size of HTR3E (approximately 51 kDa)

• Transfer and blocking:

  • PVDF membranes are commonly used for optimal protein binding

  • Block with 5% non-fat milk or BSA in TBST

• Antibody incubation:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Overnight incubation at 4°C often yields best results

  • HRP-conjugated secondary antibodies or alternative detection systems

• Detection:

  • Enhanced chemiluminescence (ECL) reagents

  • Exposure times may need optimization based on expression levels

How can researchers experimentally assess the specificity and efficacy of different HTR3E antibodies?

To evaluate HTR3E antibody quality:

• Comparative analysis:

  • Test multiple antibodies targeting different epitopes of HTR3E

  • Compare antibodies from different vendors or different clones from the same vendor

  • Evaluate batch-to-batch consistency through repeated testing

• Specificity controls:

  • Peptide competition/blocking assays using the immunizing peptide

  • Testing in HTR3E-overexpressing and knockdown/knockout models

  • Testing in tissues with differential HTR3E expression levels

• Cross-reactivity assessment:

  • Evaluate potential cross-reactivity with other HTR3 family members (HTR3A, HTR3B, HTR3C, HTR3D)

  • Consider sequence homology between species when working with non-human samples (mouse HTR3E has only 33% identity with human HTR3E)

• Application-specific validation:

  • For IHC: evaluate staining patterns and compare with known expression patterns

  • For WB: confirm single band at expected molecular weight

  • For FACS: compare with isotype controls and unstained samples

• Reproducibility testing:

  • Replicate experiments under identical and varied conditions

  • Document lot-to-lot variation when using commercial antibodies

How should researchers interpret HTR3E expression changes in disease states such as IBS-D?

When analyzing HTR3E expression in pathological conditions:

• Contextualize changes within serotonin signaling pathways:

  • Consider HTR3E changes in relation to other 5-HT receptor subtypes

  • Evaluate impacts on downstream signaling cascades

  • Assess relationships with serotonin synthesis, release, and reuptake mechanisms

• Account for genetic variation:

  • Genotype samples for known functional variants (e.g., rs56109847)

  • Stratify expression data by genotype (significant differences have been observed between GG and GA genotypes)

• Consider regional specificity:

  • HTR3E expression varies across GI regions

  • Sigmoid colon has shown significant expression changes in IBS-D patients

  • Compare multiple GI regions when possible

• Correlate with functional outcomes:

  • Link expression changes to electrophysiological measurements

  • Correlate with symptom severity in clinical samples

  • Establish relationships with other biomarkers of GI dysfunction

• Evaluate sex-specific effects:

  • HTR3E variants show stronger associations with IBS-D in females

  • Analyze male and female samples separately when possible

What experimental designs are recommended for studying miRNA regulation of HTR3E expression?

To investigate miRNA-mediated regulation of HTR3E:

• Bioinformatic analysis:

  • Identify potential miRNA binding sites in HTR3E 3'-UTR

  • Evaluate how genetic variants affect predicted miRNA binding

  • Focus on miRNAs expressed in relevant tissues (e.g., miR-510 in colonic tissue)

• Reporter assays:

  • Construct dual-luciferase reporters containing wild-type or variant HTR3E 3'-UTR

  • Co-transfect with miRNA expression plasmids or mimics

  • Measure relative luciferase activity to assess binding efficiency

• Expression correlation studies:

  • Quantify miRNA and HTR3E mRNA/protein levels in the same samples

  • Analyze inverse correlations characteristic of miRNA-mediated repression

  • Stratify by genotype to detect variant-specific effects

• Direct binding validation:

  • RNA immunoprecipitation of miRNA-RISC complexes

  • Pulldown assays using biotinylated miRNAs

  • Assess enrichment of HTR3E mRNA in precipitated complexes

• Functional manipulation:

  • Overexpress or inhibit specific miRNAs (e.g., miR-510)

  • Measure consequent changes in endogenous HTR3E expression

  • Rescue experiments by modifying the miRNA binding site in HTR3E 3'-UTR

What are the key considerations when designing studies to investigate the role of HTR3E in disease pathophysiology?

When researching HTR3E's role in disease:

• Sample selection and stratification:

  • Include adequate sample sizes for statistical power

  • Stratify by sex (female-specific associations have been reported)

  • Consider ethnicity (associations have been confirmed in various populations)

  • Carefully define disease phenotypes (e.g., IBS-D vs. mixed IBS vs. functional dyspepsia)

• Comprehensive genetic profiling:

  • Genotype multiple relevant SNPs (rs56109847/rs62625044 in HTR3E; consider related genes)

  • Account for linkage disequilibrium between variants

  • Consider haplotype analysis rather than single SNP associations

• Multi-level molecular analysis:

  • Integrate genomic, transcriptomic, and proteomic approaches

  • Assess both mRNA and protein expression

  • Include epigenetic regulation (DNA methylation, histone modifications)

• Functional validation:

  • Include electrophysiological studies of channel function

  • Assess impact on cellular signaling pathways

  • Evaluate consequences for tissue physiology (e.g., intestinal motility, secretion)

• Translational relevance:

  • Connect molecular findings to clinical symptoms

  • Consider pharmacological implications (HTR3 antagonists)

  • Develop potential biomarkers for patient stratification